Immunoglobulins must bind to a vast array of foreign molecules and thus exist in many forms. The sequence of the variable (V) region of immunoglobulin molecules varies tremendously, conferring virtually unlimited capacity to bind antigens. The constant (C) region comes in five different varieties: α, δ, ε, γ and μ, providing five different isotypes: IgA, IgD, IgE, IgG and IgM, each of which performs a different set of functions. B cells initially produce only IgM and IgD, and must be activated or induced to produce the other isoforms, such as IgE.
The course of IgE production starts with the activation of B cells. Upon activation with an antigen, B cells follow one of two differentiation pathways: they may differentiate directly into plasma cells, which are basically antibody-secreting factories, or they may give rise to germinal centers, specialized structures within lymphoid organs. In the latter, successive rounds of mutation of the V region genes is followed by expression of the gene products on the cell surface, with selection of the cells on the basis of the affinity of the mutated immunoglobulins against the antigen.
In both pathways of antigen-induced B cell differentiation, isotype switching occurs in which the C region of the immunoglobulin heavy chain changes from the joint expression of IgM and IgD on naive B cells to expression of one of the downstream isotypes such as IgE. This switching involves the replacement of upstream C regions with a downstream C region that has biologically distinct effector functions without changing the structure of the variable portion and, hence, its specificity. For IgE switching, a deletional rearrangement of the Ig heavy chain gene locus occurs, a rearrangement that joins the switch region of the μ gene, Sμ, with the corresponding region of the ε gene, Sε. This switching is minimally induced by IL-4 or IL-13, which initiates transcription through the Sε region, resulting in the synthesis of germ-line (or “sterile”) ε transcripts; that is, transcripts of the unrearranged Cε heavy genes. This IL-4 induced transcription is inhibited by IFN-γ, IFNα, and TGF-β. A second signal, normally delivered by T cells, is required for actual switch recombination leading to IgE production. The T cell signal may be replaced by monoclonal antibodies to CD40, Epstein-Barr viral infection, or hydrocortisone.
Recently, the mechanism of class switch recombination has been explained by an accessibility model, wherein the specificity of the switch gene rearrangement is determined by the modulation of switch region accessibility; that is, the opening up of the chromatin in certain areas, allowing the required protein/enzyme complexes access to the genes.
IgE antibodies are crucial immune mediators of allergic reactions, and have been shown to be responsible for the induction and maintenance of allergic symptoms. For example, the introduction of anti-IgE antibodies has been shown to interfere with IgE function, thus working to alleviate allergic symptoms. See Jardieu, Current Op. Immunol. 7:779-782 (1995), Shields et al., Int. Arch. Allergy.
Immunol. 107:308-312 (1995).
The expression of germline transcripts has been shown to precede and be essential for immunoglobulin or antibody class switch recombination in differentiating B cells. As B cells proceed from the expression of Immunoglobulin Mu (IgM) and Delta (IgD) to the downstream forms of Ig, cytokine signals help determine which germline transcripts will be produced and thus which constant region will recombine with the variable region of the expressed Ig. For example, IL-4 has been shown to induce the expression of IgE germline transcript and TGFβ1 can induce IgA.
RNAse protection assays (RPAs) are described in Berton et al, PNAS USA 86:2829 (1989); Berton et al., Int. Immunol. 4:387 (1992); Turaga et al., J. Immunol. 151:1383 (1993); and Warren et al., J. Immunol. 155:5637 (1995), all of which are expressly incorporated by reference;
It is one object of the present invention to use RNAse protection assays to screen and evaluate candidate agents for the ability to effect one or more germline transcripts. In addition, it is an object of the invention to provide for specific RNAse protection probes (RPPs) that can be used to facilitate this identification. Furthermore, it is an object of the invention to provide kits and compositions for these assays and analyses.